Compartmentation of caffeine and related purine alkaloids depends exclusively on the physical chemistry of their vacuolar complex formation with chlorogenic acids

Coffea seeds accumulate purine alkaloids (PuAs) and chlorogenic acids (CGAs) in a correlated manner: a high concentration of PuAs (mostly caffeine) accompanies a considerable accumulation of CGAs (mostly 5-caffeoylquinic acid; 5-CQA) and vice versa. Since caffeine and related PuAs per se freely penetrate cell-, tissue-, and organ-related barriers, we suggested that the physico-chemically well-characterized PuA-CGA complex is the crucial mechanism of PuA sequestration in these plant species and therefore the cause of the above-mentioned correlation. Suspension-cultured coffee (C. arabica) cells produce the complex partners in readily measurable concentrations, and were therefore an ideal system for investigating the in situ significance of complex formation. The partition of caffeine between cells and medium was studied in relation to the concentration of 5-CQA which, like the complex, is confined to the vacuole. Induction of the phenolic pathway was monitored by measuring PAL activity. To create concentration ranges of the complex partners as wide as possible, the cultures were subjected to various conditions such as the addition of a photoperiod or methyljasmonate (both stimulating secondary product formation), 2-aminoindan-2-phosphonic acid (2-AIP) (a most potent inhibitor of PAL), and exogenous caffeine. In all experimental sets, compartmentation of caffeine (and also that of theobromine) was highly correlated to the concentration of 5-CQA. In addition, inhibition of 5-CQA synthesis by 2-AIP consequently led to a reduction of caffeine biosynthesis whereas exogenous caffeine evoked the synthesis of the phenolic counterpart (5-CQA), this indicating a regulatory connection between the complex partners. When the cell cultures were transferred from 25 degrees to 10 degrees, or to 37 degrees, caffeine shifted rapidly, as expected, from the medium into the cells or vice versa. Moreover, a modelling study showed that complex formation almost fully explains the measured degree of compartmentation. Similarly, in tissues and organs of the intact coffee plant the driving force of caffeine compartmentation was also shown to be defined by the physical chemistry of the complex. Finally, all caffeine-containing plants may have evolved basically one common strategy to sequester PuAs, i.e. the vacuolar allocation of high concentrations of one or several complexing phenols.